LC Trouble Everywhere - - Chromatography Online
LC Trouble Everywhere

LCGC North America
Volume 30, Issue 1, pp. 36-41


The above example illustrates one of the challenges of UHPLC — reducing the sample volume sufficiently to avoid band broadening. Perhaps this could be accomplished if any preceding sample-preparation steps involved reconstitution of a dried extract. For example, if an evaporation-to-dryness step was followed by reconstitution in 50 L of mobile phase, this volume could be reduced to 10 L and now a 1-L injection would contain the same mass of sample as 5 L of the 50-L volume.

Because the band-broadening effect is worse for the first peak in the chromatogram, it may be possible to mitigate the problem by increasing the retention of the first peak. If you repeat the above calculations with several different k-values for the first peak, you'll see that a 5-L injection would be allowed with k = 4–5. This would likely be too long to wait (≈1 min) for the first peak for most impatient chromatographers — after all, you bought that UHPLC system so you could have short run times!

A third option to solve this problem might be to use on-column concentration. When the injection solvent is more than 20% weaker than the mobile phase, it is possible to inject large volumes of sample because the weak solvent causes the sample to accumulate at the head of the column until the injection solvent washes through. For example, if the mobile phase was 65% acetonitrile and 35% buffer, injection in ≤45% acetonitrile would be expected to allow injection volumes much larger than the 2-L injection calculated above. Thus, if the desired injection of 5 L was in mobile phase, you could dilute the sample twofold with buffer and inject 20 L of the sample (now in ≈33% acetonitrile) and put the desired sample mass on the column with less chance of unwanted band broadening.

As with the conclusion of the previous section, try one or more of these alternative solutions and see what works.

Wash Solvent Choice

A student from Buenos Aires was using LC for the analysis of a drug product. He was having problems with carryover, so he was experimenting with different autosampler wash solvents to see if he could mitigate the problem. With some wash solvents he noticed significant peak broadening for his sample, whereas others did not cause problems. Unfortunately, when he used the most effective wash solvent, 100% methanol containing 0.1% formic acid, he observed the worst peak shape. He wondered how he could select a wash solvent that would reduce carryover, yet maintain good peak shape.

One of the most popular autosamplers in use today is the needle-in-loop design. This configuration typically uses a 100-L sample loop that has the injection needle as a part of the loop. To inject, the needle is connected to the LC mobile-phase flow path with a high-pressure seal. The nice thing about this design is that, because the needle is in the flow path, 100% of the sample (up to the loop volume) is injected. However, the entire loop contents of 100 L are injected every time, even with a small sample injection. For example, if a 1-L injection is made, the loop contains 1 L of sample plus 99 L of solvent. Usually the loop is backflushed onto the column so that the sample goes onto the column first, followed by the remainder of the loop contents. If the loop is filled with mobile phase (or a weaker solvent), there is no problem; it is as if a 1-L sample loop was used. However, if the solvent in the loop is stronger than the mobile phase, the injection plug can be distorted and cause band broadening. Obviously, it is desirable to avoid injecting an excess of strong solvent so as to avoid unwanted band broadening.

So, how do we avoid injecting too much of a strong solvent? One way is to perform the wash cycle while the sample loop is in the flow path. With most LC systems, when the injection is made, the injection valve stays in the inject position during the run. If the autosampler wash cycle is performed while the injection valve is in the inject position, the remainder of the autosampler is washed with the wash solvent, but the sample loop is not. The sample loop is thoroughly flushed with mobile phase during the run, which is usually sufficient to clean it before the next injection — after all, the sample is soluble in the mobile phase, so the mobile phase should be good enough to flush the loop. If, however, the loop is washed in the load position, the wash solvent will remain in the loop when the next sample is aspirated. Thus, any remaining wash solvent will get injected with the sample. If this wash solvent is too strong, peak distortion is likely. Some autosamplers provide for the use of two wash solvents. If this is the case, the solution to the problem is simple. First, wash with the strong wash solvent in the load position to remove any residues from the sample loop, then wash with mobile phase (or a weaker solvent). Now when the sample is picked up, the remainder of the loop will be filled with a non-band-broadening solvent, so peak distortion should not occur. A final option would be to use a smaller-volume sample loop, if this is an option with your autosampler. Using a filled-loop injection with a 1-L loop volume, for example, would allow injection of 1 L of sample and because none of the wash solvent would remain in the loop, the choice of wash solvent composition would be of little concern.


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